Biochemical and functional characterization of the Tn-specific lectin fromSalvia sclarea seeds

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antig en (GalNAc alpha-O-Ser/Thr), a specific marker of many human carcinomas. Tw o-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MAL DI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycopro tein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilib rium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This va lue did not change in the pH range 2.5-8.5, indicating that SSL does not as sociate into higher order structures. Tandem mass spectrometry and methylat ion analysis of N-glycans released from SSL by hydrazinolysis indicated tha t SSL possesses 2-3 glycosylation sites occupied with the typical plant gly cans Man alpha 1-6[(Man alpha 1-3)(Xyl beta 1-2)]Man beta 1-4-GlcNAc beta 1 -4(Fuc alpha 1-3)GlcNAc and [(Man alpha 1-3/6)(Xyl beta 1-2)]Man beta 1-4-G lcNAc beta 1-4(Fuc alpha 1-3)GlcNAc. The influence of adjacent Tn structure s on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthe tic Tn glycopeptides was independent of the density of Tn structures. On th e other hand, mAb 83D4 only reacted with glycopeptides displaying two or th ree consecutive Tn structures.